While heterologous proteins are generally identical at the amino acid level, their post-translationally attached carbohydrate moieties often differ from the carbohydrate moieties found on proteins expressed in their natural host species. Thus, carbohydrate processing is specific and limiting in a wide variety of organisms including insect, yeast, mammalian, and plant cells.
The baculovirus expression vector has promoted the use of insect cells as hosts for the production of heterologous proteins (Luckow et al. (1993) Curr. Opin. Biotech. 4:564-572, Luckow et al. (1995) Protein production and processing from baculovirus expression vectors). Commercially available cassettes allow rapid generation of recombinant baculovirus vectors containing foreign genes under the control of the strong, polyhedrin promoter. This expression system is often used to produce heterologous secreted and membrane-bound glycoproteins normally of mammalian origin.
However, post-translational processing events in the secretory apparatus of insect cells yield glycoproteins with covalently-linked oligosaccharide attachments that differ significantly from those produced by mammalian cells. While mammalian cells often generate complex oligosaccharides terminating in sialic acid (SA), insect cells typically produce truncated (paucimannosidic) and hybrid structures terminating in mannose (Man) or N-acetylglucosamine (GlcNAc) (FIG. 1). The inability of insect cell lines to generate complex carbohydrates comprising sialic acid significantly limits the wider application of this expression system.
The carbohydrate composition of an attached oligosaccharide, especially sialic acid, can affect a glycoprotein's solubility, structural stability, resistance to protease degradation, biological activity, and in vivo circulation (Goochee et al. (1991) Bio/technology 9:1347-1355, Cumming et al. (1991) Glycobiology 1:115-130, Opdenakker et al. (1993) FASEB J. 7:1330, Rademacher et al. (1988) Ann. Rev. Biochem., Lis et al. (1993) Eur. J. Biochem. 218:1-27). The terminal residues of a carbohydrate are particularly important for therapeutic proteins since the final sugar moiety often controls its in vivo circulatory half-life (Cumming et al., (1991) Glycobiology 1:115-130). Glycoproteins with oligosaccharides terminating in sialic acid typically remain in circulation longer due to the presence of receptors in hepatocytes and macrophages that bind and rapidly remove structures terminating in mannose (Man), N-acetylglucosamine (GlcNAc), and galactose (Gal), from the bloodstream (Ashwell et al. (1974) Giochem. Soc. Symp. 40:117-124, Goochee et al. (1991) Bio/technology 9:1347-1355, Opdenakker et al. (1993) FASEB J. 7:1330). Unfortunately, Man and GlcNAc are the residues most commonly found on the termini of glycoproteins produced by insect cells. The presence of sialic acid can also be important to the structure and function of a glycoprotein since sialic acid is one of the few sugars that is charged at physiological pH. The sialic acid residue is often involved in biological recognition events such as protein targeting, viral infection, cell adhesion, tissue targeting, and tissue organization (Brandley et al. (1986) J. of Leukocyte bio. 40:97-111, Varki et al. (1997) FASEB 11:248-255, Goochee et al. (1991) Bio/technology 9:1347-1355, Lopez et al. (1997) Glycobiology 7:635-651, Opdenakker et al. (1993) FASEB J. 7:1330).
The composition of the attached oligosaccharide for a secreted or membrane-bound glycoprotein is dictated by the structure of the protein and by the post-translational processing events that occur in the endoplasmic reticulum and Golgi apparatus of the host cell. Since the secretory processing machinery in mammalian cells differs from that in insect cells, glycoproteins with very different carbohydrate structures are produced by these two host cells (Jarvis et al., (1995) Virology 212:500-511, Maru et al. (1996) J. Biol. Chem. 271:16294-16299, Altmann et al. (1996) Trends in Glycoscience and Glycotechnology 8:101-114). These differences in carbohydrate structure can have dramatic effects on the in vitro and in vivo properties of the resulting glycoprotein. For example, the in vitro activity of human thyrotropin (hTSH) expressed in insect cells was five times higher than the activity of the same glycoprotein produced from mammalian Chinese hamster ovary (CHO) cells (Grossman et al. (1997) Endocrinology 138:92-100). However, the in vivo activity of the insect cell-derived product was substantially lower due to its rapid clearance from injected rats. The drop in in vivo hTSH activity was linked to the absence of complex-type oligosaccharides terminating in sialic acid in the insect cell product (Grossman et al. (1997) Endocrinology 138:92-100).
N-glycosylation is highly significant to glycoprotein structure and function. In insect and mammalian cells N-glycosylation begins in the endoplasmic reticulum (ER) with the addition of the oligosaccharide, Glc3Man9GlcNAc2 onto the asparagine (Asn) residue in the consensus sequence Asn-X-Ser/Thr (Moremen, et al., (1994) Glycobiology 4:113-125, Varki et al. (1993) Glycobiology 3(2):97-130, Altmann et al. (1996) Trends in Glycoscience and Glycotechnology 8:101-114). As the glycoprotein passes through the ER and Golgi apparatus, enzymes trim and add different sugars to this N-linked glycan. These carbohydrate modification steps can differ in mammalian and insect hosts.
In mammalian cell lines, the initial trimming steps are followed by the enzyme-catalyzed addition of sugars including N-acetylglucosamine (GlcNAc), galactose (Gal), and sialic acid (SA) by the steps shown in FIG. 2, and as described in Goochee et al. (1991) Bio/technology 9:1347-1355.
In insect cells, N-linked glycans attached to heterologous and homologous glycoproteins comprise either high-mannose (Man9-5GlcNAc2) or truncated (paucimannosidic) (Man3-2GlcNAc2) oligosaccharides; occasionally comprising alpha(1,6)-fucose (FIG. 3; Jarvis et al. (1989) Mol. Cell. Biol. 9:214-223, Kuroda et al. (1990) Virology 174:418-329, Marz et al. (1995) Glycoproteins 543-563, Altmann et al. (1996) Trends in Glycoscience and Glycotechnology 8:101-114). These reports primarily directed to Sf-9 or Sf-21 cells from Spodoptera frugiperda, indicated that insect cells could trim N-linked oligosaccharides but could not elongate these trimmed structures to produce complex carbohydrates. Reports from other insect cell lines, including Tricoplusia ni (T. ni; High Five™) and Estigmena acrea (Ea-4), indicated the presence of limited levels of partially elongated hybrid (structures with one terminal Man branch and one branch with terminal Gal, GlcNAc, or another sugar; FIG. 4a) and complex (structures with two non-Man termini; FIG. 4b) N-linked oligosaccharides (Oganah et al. (1996) Bio/Technology 14:197-202, Hsu et al. (1997) J. Biol. Chem. 272:9062-9070). Low levels of GlcNAc transferase I and II (GlcNAc TI and TII), fucosyltransferase, mannosidases I and II, and Gal transferase (Gal T) have been reported in these insect cells; indicating a limited capability for production of these hybrid and complex N-linked oligosaccharides in these cells (Velardo et al. (1993) J. Biol. Chem. 268:17902-17907, Altmann et al. (1996) Trends in Glycoscience and Glycotechnology 8:101-114, van Die et al. (1996) Glycobiology 6:157-164).
However; most insect cell derived glycoproteins lack complex N-glycans. This absence may be attributed to the presence of the hexosaminidase N-acetylglucosaminidase that cleaves GlcNAc attached to the alpha(1,3) Man branch to generate paucimannosidic oligosaccharides (Licari et al. (1993) Biotech. Prog. 9:146-152, Altmann et al. (1995) J. Biol. Chem. 270:17344-17349). Chemicals have been added in an attempt to inhibit this glycosidase activity, but significant levels of paucimannosidic structures remain even in the presence of these inhibitors (Wagner et al. (1996) J. Virology 70:4103-4109).
Manipulating carbohydrate processing in insect cells has been attempted; and in mammalian cells, the expression of sialyltransferases, galactosyltransferases and other enzymes is well established in order to enhance the level of oligosaccharide attachment (see U.S. Pat. No. 5,047,335). However, in these cases, the presence of the necessary donor nucleotide substrates, most significantly the sialylation nucleotide, CMP-sialic acid, in the proper subcellular compartment has been assumed. Attempts to manipulate carbohydrate processing have been made by expressing single transferases such as N-Acetylglucosamine transferase I (GlcNAc T1), galactose transferase (GAL T), or sialyltransferase (Lee et al. (1989) J. Biol. Chem. 264:13848-13855, Wagner et al. (1996) Glycobiology 6:165-175, Jarvis et al. (1996) Nature Biotech. 14:1288-1292, Hollister et al. (1998) Glycobiology 8:473-480, Smith et al., (1990) J. Biol. Chem. 265:6225-6234, Grabenhorst et al. (1995) Eur. J. Biochem. 232:718-725). Introduction of a mammalian beta(1,4)-GalT using viral vectors (Jarvis et al. (1995) Virology 212:500-511) or stably-transformed cell lines (Hollister et al. (1998) Glycobiology 8:473-480) indicates that both approaches can enhance the extent of complex glycosylation of foreign glycoproteins expressed in insect cells. GlcNAcT1 co-expression can increase the number of recombinant glycoproteins with oligosaccharides containing GlcNAc on the Man alpha(1,3) branch (Jarvis et al. (1996) Nature Biotech. 14:1288-1292, Jarvis et al. (1995) Virology 212:500-511, Hollister et al. (1998) Glycobiology 8:473-480; Wagner et al. (1996) Glycobiology 6:165-175).
However, the production of complex carbohydrates comprising sialic acid has not been observed in these studies. Sialylation of a single recombinant protein (plasminogen) produced in baculovirus-infected insect cells has been reported (Davidson et al. (1990) Biochemistry 29:5584-5590), but findings appear to be specific to this glycoprotein. Conversely, many reports indicate the complete absence of any attached sialic acid on glycoproteins from all insect cell lines tested to date (Voss et al. (1993) Eur. J. Biochem. 217:913-919, Jarvis et al. (1995) Virology 212:500-511, Marz et al. (1995) Glycoproteins 543-563, Altmann et al. (1996) Trends in Glycoscience and Glycotechnology 8:101-114, Hsu et al. (1997) J. Biol. Chem. 272:9062-9070).
The reason for this absence of sialylated glycoproteins was initially puzzling since polysialic acid structures were obtained in Drosophila embryos (Roth et al. (1992) Science 256:673-675). However, as demonstrated herein, it is now evident that insect cell lines generate very little sialic acid as compared to mammalian CHO cells (See FIG. 16). With very little sialic acid, the insect cells cannot generate the donor nucleotide CMP-sialic acid essential for sialylation. A similar lack or limitation in donor nucleotide substrates may be observed in other eukaryotes as well. Thus, the co-expression of sialyltransferase and other transferases must be accompanied by the intracellular generation of the proper donor nucleotide substrates and the proper acceptor substrates in order for the production of sialylated and other complex glycoproteins in eukaryotes. In addition, sialic acid and CMP-sialic acid are not permeable to cells so these substrates can not be provided directly to the medium of the cultures (Bennett et al. (1981) J. Cell. Biol. 88:1-15).
The manipulation of post-translational processing is particularly relevant to biotechnology since recombinant DNA products generated in different hosts are usually identical at the amino acid level and differ only in the attached carbohydrate composition (Goochee et al. (1991) Bio/technology 9:1347-1355). Engineering carbohydrate pathways is useful to make recombinant DNA technology more versatile and expand the number of hosts that can generate particular glycoforms. This flexibility could ultimately lower biotechnology production costs since host efficiency would be the primary factor dictating which expression system is chosen rather than a host's capacity to produce a specific glycoform. Furthermore, carbohydrate engineering is useful to tailor a glycoprotein to include specific oligosaccharides that could alter biological activity, structural properties or circulatory targets. Such carbohydrate engineering efforts will provide a greater variety of recombinant glyco-products to the biotechnology industry.
Glycoproteins containing sialylated oligosaccharides would have improved in vivo circulatory half-lives that could lead to their increased utilization as vaccines and therapeutics. In particular, complex sialylated glycoproteins from insect cells would be more appropriate biological mimics of native mammalian glycoproteins in molecular recognition events in which sialic acid plays a role.
Therefore, manipulating carbohydrate processing pathways in insect and other eukaryotic cells so that the cells produce complex sialylated glycoproteins is useful for enhancing the value of heterologous expression systems and increasing the application of heterologous cell expression products as vaccines, therapeutics, and diagnostic tools; for increasing the variety of glycosylated products to be generated in heterologous hosts; and for lowering biotechnology production costs, since particular expression systems can be selected based on efficiency of production rather than the capacity to produce particular product glycoforms.